Process for preparing human G-CSF

ABSTRACT

The present invention discloses an improved process for the production of G-CSF in high yield via a high salt-induced increase in plasmid stability during the production phase.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/280,496 filed Aug. 22, 2008, which is a 371 of PCT/IN2007/000105filed Mar. 5, 2007 which claimed priority to Indian Patent ApplicationNo. 309/MUM/2006 filed Mar. 6, 2006, the contents of which areincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to an improved process for the productionof G-CSF in high yield via a high salt-induced increase in plasmidstability during the production phase.

BACKGROUND OF THE INVENTION

The cytokine Granulocyte Colony Stimulating Factor (G-CSF) treatmentsignificantly improves the quality of life among patients with severechronic neutropenia [Jones et al. JAMA 270: 1132-1133 (1993)]. The G-CSFis a potent endogenous trigger for the release of neutrophils from bonemarrow stores and for their activation for enhanced antimicrobialactivity. G-CSF has been widely evaluated in various pre-clinical modelsof acute illness, with generally promising results [Marshall J. C. Shock24: 120-9 (2005)]. Due to its proven efficacy during chemotherapycycles, the G-CSF is an important biopharmaceutical drug used inoncology. G-CSF has been cloned and expressed in various types of cells,e.g. microbial cells [Souza L. M. Science 232: 61-65 (1986); Hu Z. Y. etal. Zhongguo Shenghua Yaowu Zazhi (1999), 20: 55-57], yeast cells[Lasnik M. A. et al. Biotechnol. Bioeng. 81: 768-774 (2003); Lee S. M.et al. Korean patent KR 160934 B1 19981116], rice cells [Hong et al.Protein Expr Purif. Epub ahead of print (2005)], feline cells [Yamamotoet al. Gene 274: 263-269 (2001)], Chinese Hamster Ovary cells [Monaco L.et al. Gene. 180:145-150 (1996)], insect cells [Shinkai et al. ProteinExpr Purif. 10: 379-385 (1997)], and even in transgenic goat [Ko J. H.et al. Transgenic Res. 9: 215-22 (2000)]. For pharmaceutical use theG-CSF is produced primarily in Escherichia coli [Jevsevar S. et al.Biotechnol. Prog. 21: 632-639 (2005)], where it is produced as inclusionbodies, which are insoluble aggregates of the recombinant protein innon-native conformation [Baneyx F. & Mujacic M. Nature Biotechnol. 22:1399-1408 (2004)], that generally do not have biological activity[Bemardez C. E. Curr. Opin. Biotechnol. 9: 157-163 (1998)]. Thetechnologies of its secretory production [Jeong K. J. & Lee S. Y.Protein Expr Purif. 23: 311-318 (2001); Lee S. Y. et al. Methods Mol.Biol. 308: 31-42 (2005)], have also been reported. Secretory expressiongenerally results into the release of properly folded form of G-CSF intothe periplasmic space or extra-cellular medium, but the yields are muchlesser than those obtained with inclusion bodies. It is thereforecommercially beneficial to express G-CSF in E. coli as inclusion bodies.Properly folded, biologically active G-CSF protein is easily obtainedfrom inclusion bodies in a commercially viable manner, usingdenaturation and renaturation processes applied subsequent to theisolation and solubilization of inclusion bodies [Rudolph R, In ProteinEngineering: Principles and Practice; Cleland, J. L., Craik, S. C.,Eds.; Wiley-Liss, Inc.: New York, 1996; pp 283-298; Rathore A. S. et al.J Pharm Biomed Anal 32:1199-1211 (2003)].

One of the most efficient methods of recombinant protein production inE. coli is fed-batch, which can be carried out, in cyclic and non-cyclicmodes. The non-cyclic processes are less complex and therefore moresuitable for industrial production. In fact, prior art describes one ofthe highest GCSF yields from a non-cyclic fed-batch process which is inthe range of 4.2-4.4 g/L [Yim S C et al. Bioprocess and BiosystemsEngineering (2001), 24, 249-254]. Carrying out fed-batch fermentation incyclic mode in order to obtain higher cumulative yield results in highplasmid instability [Choi S.-J. et al. J. Microbial. Biotechnol. 10:321-326 (2000)], thereby limiting the robustness of the process.

In general, in order to have high expression of the product it isimperative to keep the product gene-containing extra-chromosomal plasmidinside the cell in its proper form. This is generally achieved bymaintaining selection pressure on the recombinant microorganism byadding a suitable antibiotic to the culture broth. Increase inexpression level of G-CSF by adding antibiotic (Ampicillin) every 1-2 hduring fermentation to decrease the ‘segregational nonstability’ ofrecombinant strain has been reported (Krivopalova G. N. et al. RussianPatent RU 2158303 C2-20001027). The regulatory requirement of theevidence of antibiotic clearance from the final product necessitates thelimit of its use. Higher usage of antibiotics may also have a higherpotential of having an undesirable environmental impact. But thedecrease in antibiotic selection pressure often results in decreasedplasmid stability and expression levels, which compromises therobustness of process. Therefore, it is a technical challenge to limitthe use of antibiotic while increasing the plasmid stability andexpression level of the product, especially during production phase.Further, low plasmid stability during production phase can also be dueto metabolic stress [Saraswat V. et al. FEMS Microbiol. Lett. 179:367-373 (1999)], and might lead to low expression levels [Cheng C. et alBiotechnol. Bioeng. 56: 23-31 (1997)], typically in high volumecultures.

Besides maintaining high antibiotic selection pressure, plasmidstability can be improved at the level of vector construction [SchwederT. et al. Appl Microbiol Biotechnol. 38:91-93 (1992); Pan S. H. andMalcom B. A. Biotechniques. 29:1234-1238 (2000)]. In process it can beimproved by adjusting the culture conditions, such as avoiding nutrientstarvation [Smith & Bidochka Can. J. Microbial. 44: 351-355 (1998)].While carrying out large-scale substrate-limiting fed-batch processes,nutrient to limitation/starvation is imminent, and adding antibioticeither too frequently or in large amounts are also impractical andexpensive solutions to maintain high product yield and high plasmidstability in a process of low complexity. Therefore, there is a clearneed to develop an alternate process for preparing G-CSF in highvolumetric yields by maintaining high plasmid stability using a simpleand robust process.

SUMMARY OF INVENTION

The present invention describes a non-cyclic fed-batch process toproduce Granulocyte Colony Stimulating Factor (G-CSF) in high volumetricyields in Escherichia coli by maintaining high plasmid stability in theculture, with the use of Potassium in combination with either Magnesiumor Sodium ions in high concentrations in the production medium andculture broth.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of using high concentration of Potassium andSodium cations in production phase on the stability of G-CSFgene-containing plasmid in BL21(DE3) cells at the time of batch harvest.Batch 2 was carried out with high concentrations of Potassium and Sodiumsalts, whereas Batch 1 was carried out without high concentrations ofPotassium and Sodium salts in production phase. The batches were carriedout separately in a 30-L fermenter.

FIG. 2 shows the effect of using high concentration of Potassium andSodium cations in production phase on G-CSF volumetric yield inharvested batches. Batch 2 was carried out with high concentrations ofPotassium and Sodium salts, whereas Batch 1 was carried out without highconcentrations of Potassium and Sodium salts in production phase. Thebatches were carried out separately in a 30-L fermenter.

FIG. 3 shows the effect of using high concentration of Magnesium andPotassium cations in production phase on the stability of G-CSFgene-containing plasmid in BL21(DE3) cells at the time of batch harvest.Batch 4 was carried out with high concentration Magnesium salt, whereasin Batch 3 high concentration of Magnesium salt was not used inproduction phase. Both batches were carried out with high concentrationof Potassium salt, separately in a 30-L fermenter.

FIG. 4 shows the effect of using high concentration of Magnesium andPotassium cations on G-CSF volumetric yield in harvested batches. Batch4 was carried out with high concentration Magnesium salt, whereas inBatch 3 high concentration of Magnesium salt was not used in productionphase. Both batches were carried out with high concentration ofPotassium salt separately in a 30-L fermenter.

FIG. 5 shows the effect of using high specific growth rate in productionphase on G-CSF volumetric yield in harvested batches. During productionphase Batch 4 had average specific growth rate of about 0.04 l/h,whereas Batch 5 had average specific growth rate of about 0.07 l/h. Bothbatches were carried out with high concentrations of Potassium andMagnesium salts, separately in a 30-L fermenter.

FIG. 6 shows the effect of using high concentration of thiamine inproduction phase on G-CSF volumetric yield in harvested batches. Duringproduction phase Batch 1 had 7 g/L thiamine in the production medium,whereas Batch 6 was done without using any thiamine in the productionmedium. Both batches were carried out separately in a 30-L fermenter.

DESCRIPTION OF THE INVENTION

The present invention relates to an improved fermentation process forthe production of Granulocyte Colony Stimulating Factor (G-CSF) atimproved levels of volumetric yield. It also discloses culturingconditions for improved plasmid stability that further leads to a highvolumetric yield of G-CSF. The process of the invention involves anon-cyclic fed-batch process via multiple inductions, carried out in thepresence of high concentration of Potassium, in combination with otherinorganic salts such as Sodium, Magnesium and the like, at highconcentrations in the production media. Surprisingly, when such aprocess using the salts of the of the present invention as ishereinafter described in details, was used in combination with highspecific growth rate, it still maintained high plasmid stability leadingto further increase of volumetric yield.

The present invention is further described in detail below:

Any Granulocyte Colony Stimulating Factor (also referred here as‘G-CSF’) polypeptide can be utilized. The term “Granulocyte ColonyStimulating Factor” or “G-CSF” refers to native G-CSF, muteins,fragments, fusions, analogs and derivatives thereof either exhibiting atleast 60% biological or receptor binding activity as the native hG-CSFor retaining at least about 80% amino acid identity. Examples of suchG-CSF sequence includes Genbank Sequence ID GI:27437048 and thosedescribed in U.S. Pat. No. 4,810,643.

Escherichia coli cells are transformed with suitable expression vectorcomprising the coding sequence of G-CSF and a suitable promotor selectedfrom t7, tac, and similar promoters along with other vector componentsusing transformation techniques well known in art.

In the process described below the fermentation refers to an aerobicgrowth of microorganisms, preferably recombinant E. coli, for theproduction of G-CSF. In such a process, the batch phase of growth refersto the period in which after inoculation, no nutrient, except ammoniumhydroxide is added (if needed) to the culture broth in the fermenter.The culture broth is a suspension of cells, media, and derivatives ofthe media and cells (if any). The substrate limiting fed-batch in growthphase refers to that part of growth phase in which major increase inbiomass (at least 2 doublings) takes place by adding the fed batchgrowth media to the culture broth in such a way that the concentrationof the main carbon/energy source (for example, glucose) is limiting. Theflow rate of the said media determines the specific growth rate of theculture. Pre-induction media refers to the media, having a differentcomposition than the growth media, which is added to the culture brothbefore adding the inducer (for example IPTG). Induction is the processof appreciably increasing the concentration of the G-CSF in the cells,as determined by tools known in the prior art, by addition of inducer(for example, IPTG and lactose). Production media has a differentcomposition than the growth media and is added to the culture broth inthe fermenter during the induction of the G-CSF gene. The productionmedia was also added in such a way that the concentration of substrate(for example, glucose) remains limiting. The flow rate of productionmedia determines the specific growth rate of the culture duringproduction phase.

The host cells of E. coli, previously transformed with a suitableexpression vector encoding G-CSF, were initially cultured at 37° C. inshaker-flasks to develop the seed for fermenter. The seed culture wasused for inoculating the sterile growth media in a fermenter. Thesubstrate-limiting fed-batch mode of growth phase of fermentation isinitiated once the recombinant E. coli culture starts to grow and theglucose concentration in the culture broth drops down to 0.5 g/L orless. The feed of fed-batch growth medium, added in substrate-limitingfed-batch manner, is kept continuous (exponential or constant rate) ordiscontinuous during growth phase. After achieving a cell density of1-60 g/L dry cell weight and a glucose concentration less than 0.5 g/Lin the culture broth, addition of the pre-production medium is done andsubsequently the feed of production media started and continued in acontinuous- or discontinuous-substrate-limiting fashion. Multipleinductions of G-CSF gene are made with IPTG. The average specific growthrate is not decreased during or after the additions of the inducer. ThepH is maintained at about 5-7. The temperature is maintained at about30-42° C. After 2 to 48 h of adding pre-production medium, the culturemedium is removed and subjected to down stream processing according tothe techniques described in art.

The media of the growth phase comprise carbon and energy sourcesselected from the group comprising of glucose, glycerol, etc. and thelike or mixtures thereof, complex media components selected from thegroup comprising of yeast extract, tryptone, peptone, casein enzymehydrolysate, soybean casein hydrolysate and the like, or mixturesthereof, suitable salts/nutrients selected from the group comprising ofcitric acid, potassium chloride, sodium chloride, magnesium sulphate,di-ammonium hydrogen phosphate, potassium dihydrogen phosphate, sodiumbutyrate, thiamine, glycine, and zinc chloride.

Other fermentation conditions like aeration, agitation, inoculum, timeof inoculation etc. are all chosen as per convenience as are known inprior art.

The pre-production medium comprises, complex media components selectedfrom yeast extract, tryptone, peptone, casein enzyme hydrolysate,soybean casein hydrolysate, along with nutrients such as thiamine,glycine and the like or mixtures thereof; antibiotics like kanamycin,and ampicillin and the like. Suitable salts are selected from the groupcomprising of citric acid, potassium chloride, sodium chloride,magnesium sulphate, di-ammonium hydrogen phosphate, potassium dihydrogenphosphate, sodium butyrate, and zinc chloride such that the mediumcontains high level of K ion concentration in combination with either Naor Mg ions.

The production medium contains a carbon source in addition to theconstituents of the pre-production medium. Suitable carbon source can beselected from the group consisting of glycerol, glucose, fructose andthe like or mixtures thereof. The preferred carbon source of the presentinvention is glucose. During production phase, the culture broth ismaintained with high level of K ions in combination with either Na or Mgions. The concentration of K ions is maintained at about 60 mM to about300 mM, Na ions at about 60 mM to about 300 mM, and Mg ions at about 150mM to about 250 mM in the culture broth. In a preferred embodiment, theK ion concentration is 90 mM to 150 mM, Na ion concentration is 60 mM to120 mM, and the Mg ion concentration in the culture broth is in therange of 180 mM to 220 mM.

In a further embodiment, the addition of thiamine in high concentration(in the range of 5 g/L to 10 g/L) provides yield of G-CSF in the rangeof 5-6 g/L.

The process of the present invention results in the production of G-CSFin high yields (5-9.5 g/L) with maintenance of high plasmid stabilitythroughout the growth and the production phase (75-90%).

EXAMPLE 1 Effect of High Concentrations of Na and K Ions on PlasmidStability and Volumetric Yield

The experiment was carried out in a 30-L fermenter. A seed culture of E.coli BL21 (DE3) cells transformed with the human G-CSF gene wasinoculated in the growth media of the following composition.

Concentration before Component inoculation KH₂PO₄ 13.3 g/L (NH₄)₂HPO₄4.0 g/L Yeast extract 1.0 g/L Glucose 10.0 g/L Citric acid 1.7 g/LMgSO₄•7H₂O 1.2 g/L Trace element solution 20.0 mL/L Kanamycin 50 mg/LTrace Metal Solution:

Component Concentration FeCl₃•6H₂O 0.162 g/L ZnCl₂•4H₂O 0.0144 g/LCoCl₂•6H₂O 0.12 g/L Na₂MoO₄•2H₂O 0.012 g/L CaCl₂•2H₂O 0.006 g/L CuCl₂1.9 g/L H₃BO₃ 0.5 g/L

Adding the following ‘fed-batch growth media’ in substrate limitingfed-batch mode brought about the major increase in biomass:

Component Concentration Glucose 700 g/L MgSO₄•7H₂O 20 g/L Trace elementsolution 20 mL/L Kanamycin 500 mg/L

In growth phase ammonium hydroxide was used as the pH regulator tomaintain the pH in the range of 6.8 to 7.0. The temperature wasmaintained at 37° C. After achieving optical density of about 50 AU (at600 nm) in Batch 2 the pre-induction media, consisting of the followingcomposition, was added in the culture broth:

Component Concentration Yeast extract 84.38 g/L Potassium chloride 75.41g/L Sodium chloride 123.13 g/L Thiamine hydrochloride 8.44 g/L

The final concentration of Potassium and Sodium cations in the culturebroth was about 120 mM and 250 mM, respectively.

The feeding of the following production media was subsequently started:

Component Concentration Glucose 270 g/L MgSO₄•7H₂O 1 g/L Yeast extract214 g/L Thiamine hydrochloride 7 g/L Potassium chloride 8.94 g/L (onlyin Batch 2) Sodium chloride 17.5 g/L (only in Batch 2)

The expression of G-CSF gene was induced by multiple additions offilter-sterilized solution of IPTG to the culture broth. In productionphase ammonium hydroxide was used as the pH regulator to maintain the pH6.8. The temperature was maintained at 37° C. Kanamycin was added to theculture to put selection pressure. The amount of Kanamycin used duringproduction phase of Batch 2 (37.5 mg, added once), was about 1% of theamount used in Batch 1 (2925 mg, multiple additions) in order to greatlychallenge the effect of salts on plasmid stability.

The plasmid stability was determined by first aseptically collecting theend-of-the-batch sample in a sterile tube and aseptically spreadingappropriate volume of the suitable diluted sample on Luria-Bertanimedium with and without Kanamycin (50 mg/L). The plates were incubatedat 37° C. for 48 hours and the plates, having statistically significantcolonies were counted. A value obtained by dividing the number ofcolonies obtained on Kanamycin-containing plates with those on Kanamycinnon-containing plates was used to calculate plasmid stability. Theplasmid stability in Batch 2 (96.8%) was more than twice, as compared tothe plasmid stability in Batch 1 (45.0%), thereby showing the importanceof Sodium and Potassium cations in improving the plasmid stability (FIG.1).

The volumetric yield of G-CSF, as determined by densitometricquantification of the GCSF band with respect to the standard plot ofauthentic standard, after SDS-PAGE, was 5.38 g/L in Batch 1 and 5.81 g/Lin Batch 2. The volumetric yield in Batch 2 was about 8% higher thanthat of Batch 1 (FIG. 2).

EXAMPLE 2 Effect of High Concentrations of Magnesium and PotassiumCations on Plasmid Stability and Volumetric Yield

The experiment was carried out in a 30-L fermenter. Since the productionphase media of both batches (Batch 3 and Batch 4) were identicalincluding the concentration of Potassium cation, except only theconcentration of Magnesium cation, the results reflected the effect ofthe combination of Potassium and Magnesium cation. A seed culture of E.coli BL21 (DE3) cells transformed with the human G-CSF gene wasinoculated in the growth media of the following composition.

Concentration before Component inoculation KH₂PO₄ 13.3 g/L (NH₄)₂HPO₄4.0 g/L Yeast extract 1.0 g/L Glucose 10.0 g/L Citric acid 1.7 g/LMgSO₄•7H₂O 1.2 g/L Trace element solution 20.0 mL/L Kanamycin 50 mg/LTrace Metal Solution:

Component Concentration FeCl₃•6H₂O 0.162 g/L ZnCl₂•4H₂O 0.0144 g/LCoCl₂•6H₂O 0.12 g/L Na₂MoO₄•2H₂O 0.012 g/L CaCl₂•2H₂O 0.006 g/L CuCl₂1.9 g/L H₃BO₃ 0.5 g/L

Adding the following ‘fed-batch growth media’ in substrate limitingfed-hatch mode brought about the major increase in biomass:

Component Concentration Glucose 700 g/L MgSO₄•7H₂O 20 g/L Trace elementsolution 20 mL/L Kanamycin 500 mg/L

In growth phase ammonium hydroxide was used as the pH regulator tomaintain the pH in the range of 6.8 to 7.0. The temperature wasmaintained at 37° C. After achieving optical density of about 50 AU (at600 nm) the pre-induction media, consisting of the followingcomposition, was added in the culture broth:

Component Concentration Yeast extract 84.38 g/L Potassium chloride 75.44g/L Thiamine hydrochloride 8.38 g/L Magnesium Sulfate 187.06 g/L (onlyin Batch 4)The feeding of the production following media was subsequently started:

Component Concentration Glucose 270 g/L Magnesium Sulfate 1 g/L (only inBatch 3) Magnesium Sulfate 50.3 g/L (only in Batch 4) Yeast extract 214g/L Thiamine hydrochloride 7 g/L Potassium chloride 8.94 g/L

The expression of G-CSF gene was induced by multiple additions offilter-sterilized solution of IPTG to the culture broth. In productionphase ammonium to hydroxide was used as the pH regulator to maintain thepH 6.8. The temperature was maintained at 37° C. Kanamycin was added tothe culture to put selection pressure. The equal amount of Kanamycin(37.5 mg, added once) was used during production phase. Theconcentration of Potassium and Magnesium cations in the culture brothduring production phase was about 120 mM and 200 mM, respectively.

The plasmid stability was determined as described previously The plasmidstability in Batch 4 (97.3%) was about 6% higher than the plasmidstability in Batch 3 (91.8%), thereby showing the effectiveness ofMagnesium and Potassium cations in improving the plasmid stability (FIG.3). The plasmid stability obtained in presence of Magnesium andPotassium cations was 116.2% higher than that in Batch 1 (without highconcentration of salts in production phase).

The volumetric yield of G-CSF, as determined by densitometricquantification of the GCSF band with respect to the standard plot ofauthentic standard, after SDS-PAGE, was 5.48 g/L in Batch 3 and 8.35 g/Lin Batch 4 (FIG. 4). The volumetric yield in Batch 4 was about 55%higher than that on Batch 1.

EXAMPLE 3 Effect of High Specific Growth Rate During Production Phase onVolumetric Yield

The experiment was carried out in a 30-L fermenter. The production phasemedia composition of both batches (Batch 4 and Batch 5) were identicalincluding the concentrations of Potassium and Magnesium cations. Theaverage specific growth rate during production phase of Batch 5 washigher than that in production phase of Batch 4. A seed culture of E.coli BL21 (DE3) cells transformed with the human G-CSF gene wasinoculated in the growth media of the following composition.

Concentration before Component inoculation KH₂PO₄ 13.3 g/L (NH₄)₂HPO₄4.0 g/L Yeast extract 1.0 g/L Glucose 10.0 g/L Citric acid 1.7 g/LMgSO₄•7H₂O 1.2 g/L Trace element solution 20.0 mL/L Kanamycin 50 mg/LTrace Metal Solution:

Component Concentration FeCl₃•6H₂O 0.162 g/L ZnCl₂•4H₂O 0.0144 g/LCoCl₂•6H₂O 0.12 g/L Na₂MoO₄•2H₂O 0.012 g/L CaCl₂•2H₂O 0.006 g/L CuCl₂1.9 g/L H₃BO₃ 0.5 g/L

Adding the following ‘fed-batch growth media’ in substrate limitingfed-batch mode brought about the major increase in biomass:

Component Concentration Glucose 700 g/L MgSO₄•7H₂O 20 g/L Trace elementsolution 20 mL/L Kanamycin 500 mg/L

In growth phase ammonium hydroxide was used as the pH regulator tomaintain the pH in the range of 6.8 to 7.0. The temperature wasmaintained at 37° C. After achieving optical density of about 50 AU (at600 nm) the pre-induction media, consisting of the followingcomposition, was added in the culture broth:

Component Concentration Yeast extract 84.38 g/L Potassium chloride 75.44g/L Thiamine hydrochloride 8.38 g/L Magnesium Sulfate 187.06 g/LThe feeding of the following production media was subsequently started:

Component Concentration Glucose 270 g/L Magnesium Sulfate 50.3 g/L Yeastextract 214 g/L Thiamine hydrochloride 7 g/l Potassium chloride 8.94 g/L

The expression of G-CSF gene was induced by multiple additions offilter-sterilized solution of IPTG to the culture broth. In productionphase ammonium hydroxide was used as the pH regulator to maintain the pH6.8. The temperature was maintained at 37° C. Kanamycin was added to theculture to put selection pressure. Equal amount of Kanamycin (37.5 mg,added once) was used during production phase. The average specificgrowth rate during production phase in Batch 4 was about 0.04 l/h,whereas that in Batch 5 was about 0.07 l/h.

The volumetric yield of G-CSF determined, as described previously, to be8.35 g/L in Batch 4 and 9.94 g/L in Batch 5. The volumetric yield inBatch 5 was about 19% higher than that on Batch 4 (FIG. 5). The plasmidstability in the end-of-the-batch samples of both batches was high(>75%).

EXAMPLE 4 Effect of Using High Concentration of Thiamine in ProductionPhase on Volumetric Yield

The experiment was carried out in a 30-L fermenter. A seed culture of E.coli BL21 (DE3) cells transformed with the human G-CSF gene wasinoculated in the growth media of the following composition.

Concentration before Component inoculation KH₂PO₄ 13.3 g/L (NH₄)₂HPO₄4.0 g/L Yeast extract 1.0 g/L Glucose 10.0 g/L Citric acid 1.7 g/LMgSO₄•7H₂O 1.2 g/L Trace element solution 20.0 mL/L Kanamycin 50 mg/L(In Batch 1) Ampicillin 100 mg/L (In Batch 6)Trace Metal Solution:

Component Concentration FeCl₃•6H₂O 0.162 g/L ZnCl₂•4H₂O 0.0144 g/LCoCl₂•6H₂O 0.12 g/L Na₂MoO₄•2H₂O 0.012 g/L CaCl₂•2H₂O 0.006 g/L CuCl₂1.9 g/L H₃BO₃ 0.5 g/L

Adding the following ‘fed-batch growth media’ in substrate limitingfed-batch mode brought about the major increase in biomass:

Component Concentration Glucose 700 g/L MgSO₄•7H₂O 20 g/L Trace elementsolution 20 mL/L Kanamycin 500 mg/L (In Batch 1) Ampicillin 50 mg/L ofculture broth at each addition only in Batch 6. Total six additions weremade during ‘fed batch growth’ phase of the batch.

In growth phase ammonium hydroxide was used as the pH regulator tomaintain the pH about 6.8. The temperature was maintained at 37° C.After achieving optical density of about 50 AU (at 600 nm) the feedingof the following production media was started:

Component Concentration Glucose 270 g/L MgSO₄•7H₂O 1 g/L Yeast extract214 g/L Thiamine hydrochloride 7 g/L (Only in Batch 1) Kanamycin 2.925 g(added multiple times in Batch 1) Ampicillin 2.689 g (added multipletimes in Batch 6)

The expression of G-CSF gene was induced by multiple additions offilter-sterilized solution of IPTG to the culture broth. In productionphase ammonium hydroxide was used as the pH regulator to maintain the pH6.8. The temperature was maintained at 37° C.

The volumetric yield of G-CSF was determined, as described previously.The volumetric yield in end-of-batch sample of Batch 1 (high thiaminebatch) was about 10.7% higher than that of in Batch 6 (4.86 g/L),thereby showing the effectiveness of high concentration of thiamine inimproving the G-CSF volumetric yield (FIG. 6).

Advantages of the Process:

-   (1) Higher volumetric yield allows the larger yield at a smaller    scale, thereby limiting the capital expenditure on scale-up.-   (2) High volumetric yield is achieved using media components    (Magnesium, Potassium, and Magnesium salts) of very low cost.-   (3) A culture having high plasmid stability is more capable of    producing volumetric yield of G-CSF in metabolic stressful    conditions, such as G-CSF gene expression in condition of high    specific growth rate.

We claim:
 1. A process for producing Granulocyte Colony StimulatingFactor (G-CSF) comprising the steps of: i) inoculating a fermenter witha suitable E. coli culture previously transformed with a suitableexpression vector encoding gene of G-CSF, ii) carrying out batch or andfed batch mode with suitable culture broth to increase biomass, iii)adding pre-induction medium to the culture broth, and iv) addingproduction medium to the culture broth in a substrate limiting fed bathmode in which multiple inductions of the nucleic acid sequence encodingG-CSF are carried out to produce G-CSF, v) wherein the culture brothcomprises at least 5 g/L thiamine, wherein the production medium has afinal concentration of potassium (K) from 60 mM to 180 mM, a finalconcentration of sodium (Na) from 60 mM to 300 mM, and a finalconcentration of magnesium (Mg) from 150 mM to 250 mM.
 2. The process asclaimed in claim 1, wherein the culture broth further comprisespotassium (K) ions in the concentration range of 90 mM to 150 mM incombination with a concentration of inorganic cations selected from theconcentration of sodium (Na) ions in the range of 90 mM to 120 mM, andmagnesium (Mg) ions in the range of 180 mM to 220 mM.
 3. The process asclaimed in claim 1, wherein the thiamine is in the range of 5 g/L to 10g/L.